Effect of Er:YAG Laser Irradiation and Acidulated Phosphate Fluoride Therapy on Re-Mineralization of White Spot Lesions

Statement of the Problem: Studies on the efficacy of erbium laser for enhancement of enamel resistance to acid attacks and its effects on fluoride uptake by the enamel are limited. Purpose: This study sought to assess and compare the effects of erbium-doped yttrium aluminum garnet (Er:YAG) laser irradiation and application of acidulated phosphate fluoride (APF) gel (alone and in combination) on remineralization of artificial white spot lesions (WSLs). Materials and Method: This in vitro, experimental study evaluated 90 buccal and lingual slabs of extracted human premolars. The specimens underwent pH cycling to induce WSLs. They were then randomly divided into 6 groups of caries-free positive control (c+), negative control with WSLs (ws), 1.23% APF gel applied on the enamel (F), Er:YAG laser irradiation (80 mJ, 10 Hz, and 8 J/cm2) of enamel (L), APF gel application followed by laser irradiation (FL), and laser irradiation followed by fluoride gel application (LF). The fluoride ion content of specimens was measured before and after the intervention using a potentiometer. Data were analyzed by ANOVA (p< 0.05). Results: APF gel application before/after laser irradiation maximally increased the fluoride uptake by the enamel (p= 0.000). Application of APF gel in group F and laser irradiation in group L increased fluoride uptake by the enamel, compared with groups 1 and 2 (p= 0.000). Laser- treated (L) and APF-treated (F) groups had no significant difference in this respect (p= 0.945). Maximum fluoride concentration was noted in combined laser and fluoride groups (FL=332.07ppm and LF=341.27ppm) with no significant difference between the two (p= 1.000). Conclusion: Er:YAG laser irradiation changes the chemical composition of enamel and probably promote its remineralization, especially when combined with APF gel application, which highlights its cariostatic potential.


Introduction
Nowadays, modern caries management focuses on noncavited carious lesions, preventing caries progression and their remineralization instead of drilling of tooth structure and filling. Recently, there have been many innovative advances in enamel remineralization promotion. Some of these methods are independent of fluoride therapy, such as tooth regeneration via dentin phosphoprotein. This material can promote remineralization of the tooth surface when it is present in a solution includ-ing calcium and phosphate, just like saliva [1][2]. Moreover, modern systems such as recombinant porcine amelogenin (rP172) and leucine-rich amelogenin peptide, containing amelogenin (the protein that regulates the growth and maturation of enamel crystals in newly formed enamel matrix) stabilize calcium phosphate to promote crystal formation and direct mineral growth [3][4]. Nano hydroxyapatite is an important bioactive material that has ability to enhance enamel remineralization.
Hence, these nano-sized particles can repair enamel surface via binding strongly to the enamel surface and filling up the holes and gaps of it [5]. However, it has been well documented that fluoride therapy could be considered as the cornerstone and best method for noninvasive caries management and remineralizing of white spot lesions (WSLs) [6][7].  [8]. Although topical application of fluoride is effective for prevention of WSLs, it is not an efficient method for treatment and control of these lesions, because deposited fluoride may be lost again in vivo by back diffusion, back exchange, and migration from the mineral to the surrounding tissue fluid, saliva, or plaque fluid [9]. Therefore, reservoir-releasing fluoride decreases after short periods. Some strategy has been suggested to overcome this problem, such as several applications of topical fluoride, or increasing fluoride uptake by laser irradiation [10].
Recently, lasers are used for caries prevention due to their significant effects on dental hard tissue [11]. Laser irradiation increases the enamel acid resistance by mechanisms such as fusion, changing the crystallinity, and decreasing the permeability of enamel to chemical agents [12][13]. Laser irradiation increases the size of hydroxyapatite crystals by melting, causes recrystallization of enamel, and subsequently decreases the permeability of enamel and enhances its resistance to acid attacks [14]. Evidence shows that erbium laser is highly capable of enamel removal, and its radiation can prevent caries development by decreasing the microorganism count and causing chemical and morphological changes in the enamel structure [15][16].
Studies on the efficacy of erbium laser for enhancement of enamel resistance to acid attacks and its effects on fluoride uptake by the enamel are limited. Thus, this study sought to assess and compare the effects of erbium-doped yttrium aluminum garnet (Er: YAG) laser and APF gel on remineralization of WSLs. WSLs. This solution was changed weekly [18]. After completion of demineralization, all specimens were visually inspected by the same operator for detection of WSLs. The specimens with surfaces that appeared normal in presence of water but showed an opaque chalky appearance in absence of water were separated and examined by DIAGNOdent (Kavo, Germany) to ensure the development of WSLs.

Materials and Method
The device was calibrated against a proprietary ceramic standard before each measurement. Scores 14-20 displayed by the device confirmed the presence of WSLs [19][20]. Next, the specimens were randomly divided into 5 groups of 15each. The negative control group (WS) was previously separated. The study groups have been shown at Table 1.

Fluoride gel application
For F (APF gel) group, APF gel (Sultan Chemist, Englewood NJ, USA) containing 1.23% fluoride was applied on the surface by a cotton roll in 1-2mm thickness for 4 min and was then washed thoroughly using deionized water for one minute to remove any visible remnants of the gel and dried [20][21].

Laser irradiation
For L (laser) group, Er:YAG laser (Key III, Kavo, Germany), with the exposure settings of 80 mJ, 10 Hz, and 8J/cm 2 , with a wavelength of 2940 nm and spot size of 0.63mm was irradiated in non-contact mode without air and water spray. Laser irradiation was done in pulsed emission mode, by fiber Optic delivery system with the sapphire tip (Ø1.0mm), pulse duration of 250-500 mSec, applied with a no. 2051 hand piece. Laser was irradiated manually for 10 s by scanning movement at 5 mm distance using a hand-made jig to scan the entire exposed enamel surface [22]. Before of use, The energy levels were calibrated by calibrating the tip of the Erbium fiber with the special proprietary tip calibration handpiece, according to the manufacturer's instructions.
The energy delivered was measured periodically with a  was quantified as such [23].
A blinded operator performed all of the study steps include specimens preparation, fluoride application, laser irradiation, the laser fluorescence readings and measuring the fluoride concentration according to the manufacturaer's instructions.
Data were analyzed using ANOVA at 95% confidence interval. Table 2 shows the minimum, maximum, mean and standard deviation of fluoride concentration in the study groups. Maximum fluoride concentration was noted in  Table 3. As shown in Table 3 and Table 4, the difference between groups C+ and WS (p= 0.997), groups F and L (p= 0.945), and groups FL and LF (p= 1.000) was not significant. However, the difference between groups C+ and WS with groups F and L and also FL and LF was significant. Groups F and L also had significant differences with groups FL and LF (p= 0.000).

Discussion
According to the yielded results, the fluoride concentration of enamel has been increased following application of APF gel and Er: YAG laser irradiation. It means that laser irradiation was as effective as fluoride gel applica-  [20]. In this study, visual examination was used along with DIAGNOdent to ensure the development of WSLs after pH cycling and in order to convert a qualitative variable to a quantitative variable and for resultantly higher validity and reliability. The specimens that showed score 14-20 were considered as having WSLs according to the manufacturer's instructions [20]. producing glucose transferase enzyme [25][26]. Fluoride also promotes enamel remineralization by participation in the enamel structure and increasing the deposition of Fluor apatite on the tooth structure [27].The former mechanism is the main remineralization mechanism in this study.
Decreased solubility and increased fluoride uptake by the enamel in L (laser group) can be due to the subablative effects of Er:YAG laser, change in chemical composition of enamel, and formation of Fluor apatite crystals [28][29]. Several theories have been suggested regarding the mechanism of increasing the enamel re- Evidence shows that use of water and air coolant during laser irradiation can negatively affect its cariesprevention efficacy because the laser energy is absorbed by water and becomes less effective [15,31]. Thus, laser was irradiated without air and water coolant in this study to achieve the desired temperature (>100°C) because the carbonate loss starts after raising the temperature to 100°C. This temperature raising has just occurred in enamel surface so the possibility of pulpal damage has not been significant.
Evidence shows an association between carbonate loss and increased enamel resistance to acid attacks [23][24][25][26][27][28][29][30][31][32]. Since carbonate has less adaptation to enamel crystals, the crystals are reoriented in a more stable and more resistant form after carbonate loss [33][34]. Moreover, the critical enamel pH is 5.5, which decreases to 4.8 following laser irradiation [12]. This finding can be interpreted by noticing the fact that irradiated laser is absorbed by some certain components, and the radiation energy is directly converted to heat. This would cause structural and chemical crystallographic changes in the enamel and increases its resistance to acid attacks [34].
The oxygen and phosphate contents of the enamel significantly increase after laser irradiation due to the increased pyrophosphate content following enamel heating [13]. After laser irradiation, the calcium (Ca) content increases, this subsequently increases the Ca/P ratio especially when higher energy densities are used. Under such circumstances, calcium is released in lower amounts following acid exposure [13].
The effects of different laser temperatures on the enamel structure have been previously studied [35][36].
Laser at 100°C to 650°C and low energy (0.3 J) causes oxidation of organic materials, converts acidulated phosphate to pyrophosphate, and results in loss of water and carbonate from the enamel structure [35][36][37]. Due to the generated heat and melting phenomenon, some compounds such as tetra calcium diphosphate monoxide, alpha tricalcium phosphate (α-TCP) and betatricalcium phosphate (β-TCP) are formed. This change in the organic composition of the enamel can lead to obstruction of prisms and decreased permeability [38].
At 650°C-1100°C, decomposition and oxidation of carbonate occur. At 1100°C, all the carbonate content is lost, and a new crystalline phase of alpha tricalcium phosphate and beta-tricalcium phosphate forms, which is resistant to demineralization [39][40].
Laser application along with fluoride treatment is a novel technique to improve fluoride uptake to the enamel structure. Different laser types have been used to serve this purpose, such as CO 2 , Diode Laser, Er:YAG and Nd:YAG laser [22,28]. It has been shown that the application of CO 2 laser or diode laser in com-bination with topical fluoridation inhibits the damage caused by acid attack [22]. CO 2 laser irradiation combined with fluoride reduces the enamel solubility in acidic conditions and increases fluoride uptake [23].
Despite the fact that Er:YAG lasers are mainly used for hard tissue ablation, limited studies have reported that use of low-energy Er:YAG laser can lead to caries prevention [15]. Liu and Hsu [41] stated that reduction in carbonate content and change in organic composition of the enamel are the main caries prevention mechanisms of Er:YAG laser, which is achieved by prevention of enamel demineralization without dental tissue ablation or morphological damage to the enamel surface. The ablation range of Er:YAG laser is between 9 to 11 J/cm 2 [42]. High laser energy in each pulse and higher fre-quency of pulses per second would result in greater enamel resistance to demineralization [43].
In this study, Er:YAG laser was used with 80 mJ energy, 10 Hz repetition rate, and 8J/cm 2 energy density in non-contact mode. Laser was irradiated from 5 mm distance using a hand-made jig in order to prevent enamel ablation. The current results were in agreement with the findings of other studies and showed an increase in fluoride concentration and micro-hardness of initially demineralized enamel surfaces following laser irradiation [13,30,44]. Moreover, it has been claimed that cavity preparation by this laser increases the resistance to demineralization and prevents secondary caries [45][46].
In this study, maximum fluoride concentration was Bevilacqua et al. [30] assessed the effect of erbium laser using spectrophotometry and atomic absorption spectrometry and concluded that Er:YAG laser with 1.8 J/cm 2 and 0.9 J/cm 2 fluencies decreased acid solubility and increased fluoride uptake. Combined use of laser and topical fluoride had a greater efficacy for prevention of enamel demineralization and lower decrease in surface micro hardness [20,23,30]. In this study, the mean concentration of fluoride in groups FL and LF was higher than in other groups, which indicates that combined therapy was more effective for enhancement of enamel resistance to acid attacks.
Use of fluoride compounds along with laser irradiation yields an enamel structure, which is more resistant to caries and decreases the unfavorable changes caused by laser irradiation, since it prevents irreparable and unfavorable alterations in the enamel structure [49][50].
It should be noted that due to the high absorption of Er:YAG laser by the water in the composition of enamel, laser irradiation causes micro-explosions and subsequent ablation. This leads to formation of an irregular enamel surface that enhances plaque accumulation.
Thus, this laser should be used in sub-ablative conditions to improve chemical alterations and decrease morphological changes of the enamel surface [30,46].

Conflicts of Interest
None declared.